NCI researchers have developed an ELISA assay for VLPs of HPV-16, based on reaction to the conformationally dependent epitopes of the L1 protein, but there seems to be some crossreactivity to other high-risk types. That is, women who are infected with HPV-18 and 31 are more likely to be positive on this assay than women infected with the low-risk HPV-6 and 11. The latter show little difference from uninfected women, so the assay does not seem to be detecting antibodies that would be directed against low-risk HPV types. In a prospective study, this assay proved to be about 90 percent accurate in measuring current or past infection with HPV-16, irrespective of cytology, with a maximum false positive of about 3 percent.

This ELISA assay for HPV-16 has been used to look at different groups with various cancers. As expected, there is a highly significant odds ratio for being positive on the ELISA and cervical cancer. However, there is also a significant odds ratio with urethral cancer, vulvar cancer, and possibly, cancer of the esophagus. The association with esophageal cancer is still controversial, in part because most studies fail to find HPV DNA in these cancers. There is well-documented evidence that BPV in conjunction with a carcinogen, is responsible for esophageal cancers in cattle. The viral DNA may be absent from human esophageal cancers because a “hit and run” mechanism is at work.

Hemagglutination Assay. To provide additional information, and to overcome some of the disadvantages of the serological assay, researchers have developed a very sensitive hemagglutination assay using BVP and VLPs. Data published about 20 years ago indicated that incubation with BPV causes agglutination of mouse red blood cells. Researchers found the same response to L1 and L1-plus-L2 VLPs, and that the response is sensitive to differing concentrations and combinations and BPV proteins. Similar results were obtained with other papillomaviruses, including CRPV and several varieties of HPV.

Further research led to the development of a sensitive hemagglutination inhibition (HI) assay, in which the presence of antibodies to papillomavirus will disrupt the viruses and prevent agglutination. The HI assay is type-specific, in that BVP antibodies will prevent hemagglutination in response to VLPs of BVP but not HPV-16, and conversely HPV-16 antibodies will give a negative response for VLPs of HPV-16 but not BVP. This assay has been used to show that rabbits immunized with disrupted VLPs were not protected from CRPV, despite high antibody titers on the ELISA test, while rabbits immunized with intact VLPs were protected. This assay should be useful in measuring natural exposure to papillomaviruses, evaluating immune response after VLP vaccination, and investigating cross-protection from heterologous types after VLP vaccination.

In response to questions from the audience, Dr. Lowy added the following: